This proposal has five interrelated goals. First, we propose to complete our characterization of human and mouse stratum corneum lipids. Second, we will pinpoint specific lipids or groups of lipids that are critical for barrier function. Third, we will ascertain which lipids regulate stratum corneum cohesion and desquamation. Fourth, we intend to delineate th metabolic capabilities of the stratum corneum. Fifth, we will analyze the pathways of stratum corneum lipid biosynthesis. We will characterize the lipids by standard analytical methods, including thin layer and gas liquid chromatography, high pressure liquid chromatography, mass spectrometry, and differential scanning calorimetry. Then, these same analytical techniques will be applied to lipid extracts of experimentally perturbed and pathological stratum corneum to determine as specifically as possible those compounds that are related to either barrier function or cohesion. Next, we will prepare in vitro recombinants of stratum corneum cells and lipids to test the importance of specific lipids for permeability function and cohesion in a controlled system. Additional model systems that we will use to test factors that control cohesion and desquamation include: coupling/uncoupling of isolated stratum corneum membrane complexes and aggregation of multilamellar liposomes. Barrier function will be assessed in vivo by measurement of transepidermal water loss and in vitro by isotope diffusion across samples mounted in permeability cells. Since we suspect that variations in permeability and cohesion are due to altered lipid thermal transitions, an important feature of this proposal is the parallel analysis of most samples by differential scanning calorimetry. As a further assay of the role of certain lipids in barrier function and cohesion, we will characterize and compare lipids obtained from living epidermis vs. stratum corneum, and those found in normal vs. diseased stratum corneum. We will also compare lipids in keratinocyte cultures undergoing various degrees of differentiation, and in cultured keratinocytes from patients with inherited disorders of cornification. This combined approach should yield broad insights into the molecular basis for both stratum corneum barrier function and desquamation.